Title

Development of internal standards for quantitative PCR by subcloning

Poster Number

17

Format

Poster Presentation

Abstract/Artist Statement

DNA adducts are covalent modifications to DNA strands that includes strand breaking, cross linking and chemical modification (oxidation, alkylation, etc.) which presumably leads to mutations and eventually cancer. The presence of DNA adducts has been used as a biomarker in biological systems for DNA damage that may eventually result in unrestricted cell growth. Polymerase Chain Reaction (PCR) can be used as an assay to demonstrate the DNA damage caused by carcinogenic compounds such as cisplatin and benzopyrene by inhibition of a target PCR product due to presence of DNA adducts. We have shown previously that a simple PCR assay can be used to amplify a fragment of the p53 gene (tumor subpressor gene) and can detect reduction in DNA damage when genistein and quercetin (chemical compounds found naturally in plants) are present to inhibit formation of adducts in DNA. In order to quantitate the amounts of DNA inhibition, competitive PCR must be used. Absolute quantitation, using competitive RT- PCR, measures the absolute amount (e.g., 5.3 x 105 copies) of a specific mRNA sequence in a sample. Dilutions of a synthetic DNA sequence (identical in sequence, but slightly shorter than the endogenous target) are added to the sample DNA which replicates and are co-amplified with the endogenous target. The PCR product from the endogenous transcript is then compared to the concentration curve created by the synthetic "competitor DNA." The synthetic DNA sequence is essentially an internal standard by which we can compare PCR amplification results to. In this project we subcloned a 1057 bp fragment of the p53 gene into the pUC 19 vector. The p53 fragment was isolated from the PHP53B clone (ATCC 57254) using restriction sites for the endonuclease Ban II. Next we will ubsert a 90 bp fragment of the human Ha-ras gene into the p53 gene sequence at a unique Nco I endonuclease site. The Ha-ras gene will be isolated from total human DNA using PCR or from ATCC clone 41028 with primers containing NcoI sites. A small amount of the this internal standard will be added to future experiments amplifying the p53 gene after being reacted with varying concentrations of carcinogenic compounds (i.e. cisplatin, benzopyrene) versus incubation with anti-adducting compounds such as genistein and quercetin. This will allow us to determine how much of the original DNA was intact.

Location

Pacific Geosciences Center

Start Date

24-4-2004 9:00 AM

End Date

24-4-2004 5:00 PM

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Apr 24th, 9:00 AM Apr 24th, 5:00 PM

Development of internal standards for quantitative PCR by subcloning

Pacific Geosciences Center

DNA adducts are covalent modifications to DNA strands that includes strand breaking, cross linking and chemical modification (oxidation, alkylation, etc.) which presumably leads to mutations and eventually cancer. The presence of DNA adducts has been used as a biomarker in biological systems for DNA damage that may eventually result in unrestricted cell growth. Polymerase Chain Reaction (PCR) can be used as an assay to demonstrate the DNA damage caused by carcinogenic compounds such as cisplatin and benzopyrene by inhibition of a target PCR product due to presence of DNA adducts. We have shown previously that a simple PCR assay can be used to amplify a fragment of the p53 gene (tumor subpressor gene) and can detect reduction in DNA damage when genistein and quercetin (chemical compounds found naturally in plants) are present to inhibit formation of adducts in DNA. In order to quantitate the amounts of DNA inhibition, competitive PCR must be used. Absolute quantitation, using competitive RT- PCR, measures the absolute amount (e.g., 5.3 x 105 copies) of a specific mRNA sequence in a sample. Dilutions of a synthetic DNA sequence (identical in sequence, but slightly shorter than the endogenous target) are added to the sample DNA which replicates and are co-amplified with the endogenous target. The PCR product from the endogenous transcript is then compared to the concentration curve created by the synthetic "competitor DNA." The synthetic DNA sequence is essentially an internal standard by which we can compare PCR amplification results to. In this project we subcloned a 1057 bp fragment of the p53 gene into the pUC 19 vector. The p53 fragment was isolated from the PHP53B clone (ATCC 57254) using restriction sites for the endonuclease Ban II. Next we will ubsert a 90 bp fragment of the human Ha-ras gene into the p53 gene sequence at a unique Nco I endonuclease site. The Ha-ras gene will be isolated from total human DNA using PCR or from ATCC clone 41028 with primers containing NcoI sites. A small amount of the this internal standard will be added to future experiments amplifying the p53 gene after being reacted with varying concentrations of carcinogenic compounds (i.e. cisplatin, benzopyrene) versus incubation with anti-adducting compounds such as genistein and quercetin. This will allow us to determine how much of the original DNA was intact.